JPH11225795A - Cell injury marker - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a reagent for measuring the cell injury and cytotoxicity as a marker, etc. for the cell injury due to an ischemic injury etc. by using a reagent for measuring a phosphoglyceric acid mutase(PGAM) or its isozyme. SOLUTION: A reagent for measuring a phosphoglyceric acid mutase(PGAM) containing an enolase, a pyruvate kinase, NADH and a lactate dehydrogenase or its isozyme is used to obtain a reagent for measuring the cell injury and/or cytotoxicity caused by anoxia such as an ischemic injury. Furthermore, the isozyme of the PGAM is B type isozyme or M type isozyme. When a reagent for measuring the B type isozyme is used, an inhibitor of the PGAM such as potassium tetrathionate or sodium tetrathionate or both are contained to measure the PGAM isozyme by a rate assay method. Thereby, the cell injury or cytotoxicity or both are measured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a system for measuring cell damage and / or cytotoxicity, and can be used for judging and measuring cell damage due to, for example, ischemic damage.

[0002]

2. Description of the Related Art Phosphoglycerate mutase (Phosphog)
lyceric acid mutase (sometimes called PGAM):
EC 5.4.2.1) is one of glycolytic enzymes.
-Bisphosphoglycerate (2,3-bisphosphoglycerate
: Glycerate-2,3-P2) in the presence of 2-phosphoglycerate (Glycerate-2-P) and 3-phosphoglycerate (3-phosphoglycerate: Glycerate-3)
-P) catalyze the interchange.

[0003] There are two genes encoding PGAM in mammals. The gene for the M subunit (molecular weight: about 30,000) is expressed in adult skeletal muscle and cardiac muscle, and in these tissues, MM homodimers (M-type PGAM, M-PGAM,
GAM-MM, PGAM-M or M-type isozyme). The gene for the B subunit (molecular weight of about 30,000) is expressed in adult brain, liver, kidney and erythrocytes, and in these tissues BB homodimer (B-type PGAM, B-type
PGAM, PGAM-BB, PGAM-B or B-type isozymes). Therefore, M-type PGAM
Is classified as a muscle-specific isozyme, and B-type PGAM is classified as a non-muscle or brain isozyme. The genes of both M and B subunits are specifically expressed in myocardium. In this tissue, in addition to B-type and M-type PGAM, MB heterodimer (MB-type PGAM, MB-PGA)
M, PGAM-MB or MB type isozyme)
Also exists.

Yates et al. Have shown that the PGAM activity in normal plasma is exclusively derived from the B-type isozyme by using isoelectric focusing, which allows the differential determination of PGAM isozyme. In addition, Markert states that in normal human fetal and adult brain, mainly B-type isozymes of PGAM exist,
MB-type and M-type isozymes are found in brain tumor tissues, and their expression levels correlate with tumor malignancy, as opposed to the muscle-specific enzyme creatine kinase (Crea
tine kinase: CK) showed no such changes. However, to date, little research has been conducted on the use of PGAM and its isozymes.
The use of B-type PGAM as a stroke marker (serum sample) reported by the present inventors for a patent has been reported (JP-A-8-322595). Further, there is no report on the relationship between PGAM and the amount of oxygen at the cellular level as in the present invention, and nothing is known about its use as a marker for cytotoxicity or cytotoxicity.

[0005]

It is known that cell damage can be caused by hypoxia such as ischemia and various conditions such as pH and temperature. Also, there is a strong demand for the development of a system for quick measurement.

[0006]

SUMMARY OF THE INVENTION The present invention has been made for the purpose of responding to the above-mentioned demands in the art, and as a result of research from various fields, it has been found that PGA in a cell culture medium under a low oxygen condition.
For the first time, it was found that the M value increased, and the PG
In the measurement of AM, in addition to the measurement of total PGAM, it is possible to measure the hypoxic state by measuring the isozyme, not only to determine the degree of cell damage, but also to determine the degree of cell damage due to changes in various conditions. The new and useful knowledge was obtained, and further research was carried out.

That is, the present inventors consider that PGAM (all P
GAM, B-type PGAM, and M-type PGAM) have not only been demonstrated to be useful as cell injury markers or cell toxicity markers, but also by measuring PGAM in cells and cell cultures, Changes in oxygen status and other conditions and the degree of injury due to the change can be determined. Further, as a method for measuring PGAM and its isozymes, a method previously developed by the present inventors (Japanese Unexamined Patent Application Publication No.
322595) was confirmed to be applicable, and the present invention has been completed.

Hereinafter, the present invention will be described in detail. In the present invention, when cells are damaged by changes in factors such as oxygen, pH, temperature, and various drugs, these factors are collectively referred to as cytotoxicity. Therefore, the cytotoxic marker is indirectly a cytotoxic marker, and they are two sides of the same coin. In the present invention, oxygen refers to a state of less than oxygen content in the atmosphere to anoxic state.

First, the method for measuring PGAM according to the present invention will be described for a B-type isozyme. A method for measuring B-type PGAM is to treat a sample with a PGAM inhibitor to inactivate a specific isozyme,
It consists of measuring GAM activity. For example, when tetrathioic acid is used as a PGAM inhibitor, the M-type isozyme is almost 100% inactivated, the MB type is inactivated by about 50%, and the B-type is hardly inactivated. Therefore, the B-type isozyme can be fractionated and quantified by adding tetrathioic acid to the serum to inactivate the M-type isozyme and then measuring the remaining PGAM activity.

[0010] According to the inhibition experiment using purified PGAM isozyme, only 50% of MB type is inactivated by tetrathioic acid treatment, and 50% of the MB type remains active. Since most of the PGAM activity in tissues is due to the B-type isozyme, the residual activity of the MB-type isozyme is
This can be ignored.

As the PGAM inhibitor, any substance that can selectively inhibit the activity of PGAM or its isozyme, such as an oxidizing agent and an SH reagent, can be used. Non-limiting examples include polythionic acid and / or derivatives thereof.
As the polythionic acid, any of tri to hexathionic acids can be used, and as a derivative thereof, a potassium salt, a sodium salt or the like can be used. As a preferable example, potassium tetrathionate is exemplified.

The measuring method according to the present invention is characterized in that PGAM-M is inactivated by allowing an inhibitor such as potassium tetrathionate to act on a sample (the activity of PGAM-MB is ignored in the present biological measurement system as described above). Can be measured), and the remaining PGAM-B is measured using a measurement reagent.

The principle of the assay is as shown in Table 1 below. After inactivating the M-type isozyme using potassium tetrathionate, which is an inhibitor of the M-type PGAM isozyme, the activity is measured as follows. Is performed. That is, 2,3-bisphosphoglycerate (Gl
In the presence of ycerate-2,3-P2), 3
- a phosphoglycerate (Glycerate-3-P) 2-phosphoglycerate (Glycerate-2-P), which is referred to as PEP and H ₂ O by enolase. The PEP is then
Pyruvate is converted into pyruvate and ATP by pyruvate kinase (PK) in the presence of P, and lactate dehydrogenase (LD) is added to the resulting pyruvate in the presence of NADH.
H) to act and reduce the decrease in NADH by a rate assay (ra
te assay).

[0014]

[Table 1]

[0015] The reduction of NADH can be easily measured with commercially available measuring instruments, and PGAM-B can be measured accurately and quickly (as is all PGAM). Moreover, as is clear from the examples described later, the PGAM value in the cell culture solution sample is higher in the culture solution of cells cultured in a low oxygen state than in the high oxygen state, and this phenomenon is widely observed in various cells. PGAM that is recognized and easy to measure in each cell (total PGAM, B-type PGAM, M
Type PGAM or other isozymes) and its value can be measured. Therefore, it was found that the measurement of PGAM makes it possible to measure the hypoxic state (ischemic etc.) of cells, and PGAM is a cytotoxic marker. In particular, it has been demonstrated that it is extremely suitable as a damage marker for cells suffering from ischemic injury or hypoxic injury. Such a thing is not known at all, and PGAM is recognized as a novel cytotoxic marker. Further, according to the present invention, since cytotoxicity due to various cytotoxicities other than hypoxia can be measured, it can be used as an extremely widespread cytotoxicity marker. In other words, this marker can also be used as a cytotoxicity marker. it can.

Further, according to the present invention, it contains enolase, pyruvate kinase (PK), NADH, lactate dehydrogenase (LDH), PGAM inhibitor (potassium tetrathionate, etc .: only in the case of PGAM-B measurement), Further, if necessary, a PGAM-B measurement reagent containing a substrate and a buffer can be provided, which can be used not only as a reagent for measuring hypoxia but also as a reagent for measuring cytotoxicity other than hypoxia. Can also be used. This reagent is
As exemplified in Examples described later, it is also possible to market the reagents 1 and 2 as kits.

Although the measurement of PGAM-B has been described above, all PGAMs (sometimes simply PGAMs or T-PGAMs) show the same behavior as the B-type isozyme.
Just like PGAM-B, T-PGAM and PGAM-M also measure hypoxia, measure other physical, chemical, and biological factors that have harmful effects on cells (ie, measure cytotoxicity). It can be used as a marker for damage and / or cytotoxicity.

The method of measuring T-PGAM is exactly the same as that of the B-type isozyme except that no inhibitor is used (the principle of measurement is also the same as that of the inhibitor shown in Table 1). Except for (1), and using P in (2)
Except that GAM-B was replaced by T-PGAM), the reduction of NADH may be measured by a rate assay, and T-PGAM was determined as in the case of the B-type isozyme.
The measurement of AM can also be used to measure the hypoxic state of cells and other cytotoxicity. In this case, the measurement can be performed without using an inhibitor. For the M-type isozyme, the value can be obtained by subtracting the B-PGAM value from the T-PGAM value.

The T-PGAM assay reagent and the kit are exactly the same as the B-type isozyme except for the use of an inhibitor.
These can also be used very effectively as reagents for measuring hypoxia or cytotoxicity (toxicity). M-
The same applies to the PGAM measurement reagent and the same kit.

In practicing the present invention, a cell culture and / or
Alternatively, PGAM may be measured for the ground cell product or a separated solution thereof according to the method described above, for example, as follows.

(Culture of Smooth Muscle Cells) Male 8-10 weeks old S
Explant method from HRSP thoracic aorta
method), and cultured in a DMEM medium containing 10% fetal bovine serum. (Culture of astrocytes) Trypsin digestion method from embryonic 15-day-old SHRSP rat brain
The cultured astrocytes are collected according to od) and cultured in a DMEM medium containing 10% fetal bovine serum.

(Hypoxic Experiment) These cells were
~ 1%, preferably 12-6 at 0.1% fetal calf serum.
About 0 hours (depending on cells, usually 48 hours)
After culturing, under various oxygen concentration atmospheres (for example, under 20% oxygen or 1% oxygen) for a certain period (for example, 2, 4, or 8 days)
Culture to obtain a culture solution. For these cultures, PG
The AM is measured. In that case, the same PGAM may be measured for any of the culture solutions. For example, in the case of a brain-derived cell solution such as an astrocyte culture solution, B-type isozyme is measured. Suitable P
GAM and its isozyme type may be selected. Hereinafter, examples of the present invention will be described.

[0023]

Example 1 Smooth muscle cells were collected from the thoracic aorta of male SHRSP aged 8 to 10 weeks by the explant method, and cultured to prepare cultured smooth muscle cells.

(Explant Procedure) (1) The dissection tool was placed in a clean bench, and PBS was dispensed into a 60 mm petri dish. (2) After the rats were anesthetized with pentobarbital, the abdomen was shaved and then placed on their back on a TMS sheet. (3) After blood removal from the abdominal aorta (blood collection with a syringe),
The thoracic cavity was opened and the aorta was harvested from above the diaphragm toward the aortic arch and transferred to a 60 mm Petri dish containing PBS.

(Intra-Bench Procedure) (4) Loose connective tissue of the aortic adventitia was removed with tweezers, and the aorta was incised longitudinally with small scissors. (5) After washing off the adhered blood and the like, it was transferred to a 15 ml test tube containing 5 ml of collagenase (1 mg / ml) solution and incubated for 20 minutes in a CO ₂ incubator (SP, SR: 25 minutes, WKY: About 35 minutes). (6) After completion of the incubation, the aorta was transferred to a Petri dish containing PBS and washed lightly. The aorta was transferred toward the lid of the Petri dish and opened with the lumen up. A cotton swab was soaked in PBS to gently rub the intimal surface to remove the remaining endothelium. A light cut was made at one end with a scalpel, and the inner media was detached from the outer membrane with a clock forceps from the cut (this operation had to be done quickly so as not to dry the tissue).

(7) The media tissue was cut into pieces of about 1 mm square with a scalpel (this work also had to be done quickly so as not to dry the tissue.) (8) A 26G needle was placed on the tip of a cotton swab. Spread each piece of tissue, 25c
Glued to the bottom of the m ² flask. At that time, about 30 pieces of tissue were attached to one flask (this work was also performed quickly). (9) 5 ml of DME so that the medium does not cover the tissue piece
MF (+) medium was added. (10) The flask was capped and placed in a CO ₂ incubator in a standing state in order to adhere the tissue piece to the flask surface. After the pieces were adhered (after 10-20 minutes; less PBS around the pieces was possible), the flask was gently laid down and the pieces were soaked in the medium (care was taken not to dry the pieces). (11) It was allowed to stand for 5 to 7 days, and the state of migration was observed. After migration, the medium was replaced, and the medium was left still. (12) After a sufficient amount of cells have migrated and propagated, subculture (pa
ssage) (tissue pieces should be removed as much as possible, but may remain).

[0027]

Example 2 The cultured cells prepared in Example 1 (cells passed for 5 to 10 passages) were cultured in 0.1% fetal bovine serum for 48 hours, and then cultured under an atmosphere of 20% oxygen or 1% oxygen. 2,
After culturing for 4 to 8 days, a culture solution was obtained and used as a sample.
Table 2 below shows these samples (n = 6 for each group).
All PGAM (T-PGA)
M) Values were measured using a Hitachi 7150 automatic analyzer.

[0028]

[Table 2]

Reagents 1 (R-1) and 2 (R-2) having the following compositions were used as measuring reagents.

(Reagent 1): R-1 (ml) TEA buffer (0.1 mol / l, pH 7.6) 40.32 MgSO ₄ (0.1 mol / l) 0.54 NADH (14 mmol / l) 0 .90 ADP (21 mmol / l) 1.80 G-2,3-P2 (7 mmol / l) 0.90 LDH (5 mg protein / ml) 0.18 PK (2 mg protein / ml) 0.18 Enolase (10 mg protein / Ml) 0.18 (total: 45.00 ml)

(Reagent 2): R-2 (ml) G-3-P (95 mmol / l) 3.00 TEA buffer (0.1 mol / l, pH 7.6) 5.20 (total: 8.20 ml) )

[0032] Reagent 1 (R-1) (250 µl) and a culture solution sample (5 µl) were mixed and allowed to stand for 5 minutes. Then reagent 2
The activity of PGAM was measured using (R-2). Reagent 2
Since (R-2) contains glyceric acid-3-phosphate (substrate), the reaction was started by adding 41 μl of reagent 2 (substrate start). The measurement was started about 1.5 minutes after the addition of Reagent 2.

Using a Hitachi 7150 automatic analyzer, the assay code was set to RATE-A: 32-39, the measurement temperature was set to 37 ° C., and the decrease in absorbance A 340 nm due to the decrease in NADH for about 1.5 minutes was measured. . The measurement was performed at a sub wavelength of 405 nm. The results obtained are shown in Table 3 below.
Shown in At the same time, the value of a conventionally known cytotoxic marker LDH (lactate dehydrogenase) is also shown.

[0034]

[Table 3]

As is clear from the results of the cytotoxicity test using hypoxic exposure using the cultured SHRSP-derived cultured smooth muscle cells, the total PGAM value (U / L
/ Mg protein) is higher than in high oxygen exposure,
It became clear that M could be used as a marker in cytotoxicity, and it was confirmed that M was a marker with higher sensitivity than LDH.

[0036]

Example 3 Cultured astrocytes were collected from a 15-day-old SHRSP rat brain by trypsin digestion.
This was cultured to prepare cultured astrocyte cells.

(Under alcohol disinfection) (1) Brains of embryonic 15 to 22 day old rats were taken out. (2) Put in PBS (-) in a Petri dish.

(Process in Clean Bench) (3) The hard film was peeled off. After cleaning the brain, it was minced. (4) The petri dishes were replaced about three times in the process of shredding and cleaned. (5) Antibiotics (streptomycin and penicillin)
Was added about 10 times that of normal use and left for 5 minutes (2
Times). (6) The slice was treated with 0.25% trypsin (37
° C, 5-7 minutes). (7) Using a Pasteur pipette, pipetting was performed to the extent that the tissue piece collapsed (10 to 20 times). (8) The pellet was collected by centrifugation at 12,000 rpm for 5 minutes.

(9) DMEM + 10% FCS medium was added to stop the trypsin reaction. (10) The pellet was collected by centrifugation at 12,000 rpm for 5 minutes. (11) Each brain was cultured in a 25 cm ² flask with 6 ml of medium.

[0040]

Example 4 Prior to the measurement of B-PGAM in the above cell culture, potassium tetrathionate PGAM-B
And the effects on PGAM-M were examined by automated analysis. As samples, B-type and M-type standards (both based on pooled serum) were used and diluted with physiological saline to obtain 1
/ 1 to 1/8 samples were prepared. After various concentrations of potassium tetrathionate were added to these samples and reacted for 5 minutes, the amounts of B-type and M-type PGAM were measured using an automatic analysis method.

Using the reagent of Example 2, the amounts of PGAM-B and PGAM-M were measured by the automatic analysis method, and the results in Table 4 below were obtained. As is clear from the results, it was found that potassium tetrathionate specifically inactivates only the M-type isozyme and the B-type isozyme is not affected at all.

[0042]

[Table 4]

[0043]

Example 5 After culturing the cultured cells prepared in Example 3 (cells passed for 5 to 10 passages) in 0.1% fetal bovine serum for 48 hours, the cells were cultured under 20% oxygen or 1% oxygen atmosphere. 2,
After culturing for 4 to 8 days, a culture solution was obtained and used as a sample.

For this sample, PGA was prepared using potassium tetrathionate as an M-type PGAM isozyme inhibitor.
M activity was measured. As the measurement reagent, the PGAM activity measurement reagent (R-1) used in Example 1 was 2.25 m.
M (final concentration: 1.9 mM) was added and dissolved by dissolving potassium tetrathionate (molecular weight: 302.4) to dissolve (R-1) 15 mg of potassium tetrathionate in 22 ml of the reagent) to inactivate the M-form. A reagent for measuring B-type PGAM activity was prepared.

250 μl of a solution obtained by adding potassium tetrathionate to Reagent 1 (R-1) and 5 μl of the sample were mixed and allowed to stand for 5 minutes. In the meantime, M-type (and MB-type) PGAM in the sample is inactivated and inhibited, so that the B-type PGAM value (U / L / mg protein) is measured using reagent 2 (R-2). Table 5 shows the obtained results.

[0046]

[Table 5]

As is evident from the results of the cytotoxicity test using hypoxic exposure using the cultured astrocyte cells derived from SHRSP, B-PGAM
The value was higher than that in the case of high oxygen exposure, which revealed that B-PGAM could also be used as a marker in cytotoxicity. In addition, the measurement of LDH was attempted, but it was too low and could not be used as a marker.

[0048]

According to the present invention, PGAM (T-PGA)
M, B-PGAM and other isozymes) can be measured to measure hypoxia, and thus to measure cell damage, especially damage due to hypoxia such as ischemic damage. In addition, PGAM can be widely used as a damage marker for various cells.
Further, according to the present invention, cytotoxicity caused by various cytotoxicities other than hypoxia can be widely measured, and can be used as a wide range of cytotoxicity markers (in other words, cytotoxicity markers).

Continued on the front page (72) Inventor Yoshiyuki Taniguchi 3-6-1, Shozuzawa, Itabashi-ku, Tokyo Oriental Yeast Industry Co., Ltd. (72) Inventor Yushi Matsuo 3-6-1, Shozuzawa, Itabashi-ku, Tokyo Oriental Yeast Industry Co., Ltd.

Claims (10)

[Claims] 1. The method of claim 1, wherein the phosphoglycerate mutase (PGA)
M) or a reagent for measuring cytotoxicity and / or cytotoxicity, comprising a reagent for measuring isozymes or the isozymes thereof. 2. The reagent according to claim 1, wherein the PGAM isozyme is a B-type or M-type isozyme. 3. The reagent according to claim 1, wherein the reagent for measuring PGAM contains enolase, pyruvate kinase, NADH, and lactate dehydrogenase. 4. The reagent according to claim 1, wherein the reagent for measuring B-type isozyme contains a PGAM inhibitor. 5. The reagent according to claim 4, wherein the PGAM inhibitor is polythionic acid and / or a derivative thereof. 6. The reagent according to claim 5, wherein the PGAM inhibitor is a potassium salt and / or a sodium salt of tetrathioic acid. 7. A method for measuring cytotoxicity and / or cytotoxicity, comprising measuring PGAM in a sample by a rate assay using the reagent according to any one of claims 1 to 3. 8. A method for measuring cytotoxicity and / or cytotoxicity, comprising treating a material with a PGAM inhibitor and then measuring a PGAM isozyme by a rate assay. 9. A cytotoxic or cytotoxic marker comprising PGAM or its isozyme. 10. The marker for cytotoxicity or cytotoxicity according to claim 9, wherein the cytotoxicity is cytotoxicity caused by hypoxia.